About 40% of the commercially available enzymes are derived from filamentous fungi. Lowe, Handbook of Applied Mycology. Fungal Biotechnology (eds.) Arora, D. K. Elander, R. P. & Mukerji, K. G. 681-708 (Marcel Dekker, New York; 1992). These enzymes are usually produced by species of the genera Aspergillus and Trichoderma. Because they secrete large amounts of protein into the medium, they can be grown in large-scale fermentation, and they are generally accepted as safe for the food industry.
General problems associated with the commercial cultivation of mushrooms (Agaricus bisporus) include diseases caused by pathogens like Verticillium fungicola (dry bubble), Trichoderma aggressivum (green mold), Pseudomonas tolaasii (blotch), and dsRNA viruses (La France disease and patch disease), the major insect pest [sciarid fly (Lycoriella mali)], an extremely short shelf life of the product related to bacterial spoilage and rapid senescence, and browning (bruising) of the fruit body associated with the action of endogenous poly-phenoloxidases (PPO, like tyrosinase). To further improve product quality, conventional breeding programs for A. bisporus have been only moderately successful and may not be sufficient in the long term. This is because conventional breeding techniques for fungi are highly time consuming, and because the genetic variation in commercially available strains is limited, offering little advancement by selection (Horgen et al. “Homology between mitochondrial DNA of A. bisporus and an internal portion of a linear mitochondrial plasmid of Agaricus bitorquis” Curr Genet. 1991. 19:495-502).
In the case of A. bisporus, the main obstacle to effective breeding strategies is the rather abnormal life cycle involving the unusual simultaneous segregation of either parental nucleus into one basidiospore. After outgrowth of this basidiospore, heterokaryotic mycelium is formed containing nuclei and genetic characteristics that do not differ from those present in the parental mycelium. In addition, only little recombinational activity is observed during meiosis (Summerbell et al. Genetics, October 123(2) 1989 pp. 293-300).
To overcome the limitations of conventional breeding, investigators all over the world have attempted to develop and improve transformation methods for commercial mushrooms, such as A. bisporus, for the introduction of novel characteristics. For other fungi, as well as plants, animals, and bacteria, the application of gene transfer technology is quite common and has already resulted in commercial application.
In order to enhance the economies of protein production in microorganisms, such as fungi, there have been substantial efforts to improve the efficiency of transcription and translation, maximize the proportion of total protein directed to production of the desired product, enhance the viability of the modified host, and improve the efficiency with which the modified host may be obtained. The primary promoter used in fungal transformation to date is the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter. Using strong promoters to express heterologous genes in appropriate hosts is a major strategy in biotechnological applications. The gpd promoter is a strong promoter that can be induced by any carbon source and has been widely used in the expression of heterologous proteins in Saccharomyces cerevisiae, Pichia pastoris and other yeasts.
The gpd genes have also been cloned from basidiomycetous fungi, including Schizophyllum commune, Phanerochaete chrysosporium, Agaricus bisporus (Harmsen et al., 1992), and Lentinula edodes (Hirano et al., 1999). Among these mushrooms, genetic transformation using homologous gpd promoter was reported to be successful only in A. bisporus, Flammulina velutipes and L. edodes (Hirano et al., 2000, Kuo et al., 2004, van de Rhee et al., 1996). Although heterologous promoters have been used for the expression of drug-resistant marker genes, the genetic transformation is not sufficient to express heterologous genes (Ruiz-Diez, 2002). To sufficiently and effectively express a heterologous gene, it is important for a host cell to recognize the promoter sequence by its transcriptional machinery. Chun-Yi Kuo et al. demonstrated that a heterologous gene, hygromycin B phosphotransferase gene (hpt), can be expressed in F. velulipes (Kuo et al., 2004). However, it was found that although the gpd genes in basidiomycetous fungi are highly similar, these gene differ significantly in their promoter regions.
As illustrated by the foregoing, there is a continuing need in the art for development of effective, convenient, and expeditious fungal transformation systems and gene expression components.
It is thus an object of the present invention to provide a transformation system and in particular a strong regulatory element for fungi that will accomplish the foregoing need.
A further object of this invention is to provide mechanisms for application of transgenic techniques such as those applied to bacteria, non-filamentous fungi (yeast), plants, and animals to increase yield, disease, and pest resistance, product quality, shelf life, or culinary, nutritional, or medicinal value, to produce heterologous proteins commercially, or other such protocols.
It is yet another object of the invention to provide regulatory elements capable of driving high level protein accumulation of operably linked sequences in the fruit body of fungi, as well as tissues in plant, or animal cells.
It is yet another object of the invention to provide regulatory elements polynucleotide constructs, vectors, and transformed cells for use in such transgenic protocols.
Other objects of the invention will become apparent from the description of the invention that follows.